1. Field of the Invention
The invention relates to the field of chemical biosensors, specifically the use of electrochemical aptamer biosensors utilized in an automated in situ test for the presence of Salmonella enterica bacteria.
2. Description of the Prior Art
Salmonella is a genus of rod-shaped, gram-negative, non-spore forming, and predominantly motile enterobacteria. Salmonellae are a significant cause of food borne illness worldwide. Around 1.4 million cases of salmonellosis are reported annually in the US, with approximately 16,000 hospitalizations and 550 deaths. Salmonella alone is associated with 26% of all the food borne diarrheal cases leading to hospitalization. Salmonella bacteria are especially dangerous to humans because of their zoonotic nature, meaning that they have the ability to infect across several species.
Enteritis Salmonella (e.g. Salmonella enterica) can cause diarrhea, which usually does not require antibiotic treatment. But people at risk such as infants, HIV patients, small children, the elderly, and those with suppressed immunity can become seriously ill. Osteomyelitis may develop in children with sickle cell anemia who are infected with Salmonella. Salmonella bacteria are capable of causing typhoid fever. This infects over 16 million people worldwide each year, with 500,000 to 600,000 of these cases proving to be fatal, according to the World Health Organization.
Salmonella can survive for weeks outside a living body. Ultraviolet radiation and heat accelerate their demise; they perish after being heated to 55° C. (131° F.) for one hour, or to 60° C. (140° F.) for half an hour. They have been found in dried excrement after over 2.5 years. To protect the population from Salmonella infection, governments and other rule-making bodies have enacted many rules regarding the handling of food. For cooking at home, it is recommended that food be heated for at least ten minutes at 75° C. (167° F.) at the center of the food that is being prepared. Salmonella is not destroyed by freezing.
There have been many attempts to control the spread of Salmonella bacteria in the food supply. One method of this is to disseminate information on proper food handling and cooking techniques. This is done by a wide variety of rules and regulations regarding the production, shipping, and handling of food.
One aspect of food regulation is determining acceptable levels of Salmonella bacteria in food products. The USFDA has, for example, set an acceptable level for Salmonella in the water supply as not greater than 3CFU/4 gm. (www.fda.gov.)
Of particular concern is salmonellosis caused by multidrug resistant (MDR) strains such as Salmonella enterica serovar Typhimurium DT104 or S. enterica serovar Newport. Drug resistant strains are, by their nature, much more difficult to treat than other strains of Salmonella. They can be particularly devastating to at-risk groups, such as infants and the elderly. It is in the case of MDR strains of Salmonella especially that it is important to have accurate, easy to administer testing of food sources. In this way, the initial transmission of the pathogen to humans can be reduced or eliminated.
Because of the great need for accurate testing for the presence of Salmonella, there are many testing methods available today commercially. The USFDA has guidelines for testing (see USFDA Setting a Risk Threshold for Enteric Diseases in Drinking Water), as has the USDA (see Salmonella Testing). Testing is traditionally accomplished either through DNA based methods (e.g. GENE-TRAK Colorimetric, and TAQMAN by PE Applied Biosystems), through Immunoassay based methods (e.g. EIA Foss by Foss Electric), through immuno-latex aggulation based methods (e.g. Spectate by May & Baker Diagnostics Ltd.), and also sometimes through other biochemical methods such as a motility detection system (e.g. Salmonella Rapid Test by Oxoid).
These tests are widely used and accurate, but some can take many days to accomplish, and many of these tests are not highly automated, namely they all rely on the technician to determine the outcome of the test. Additionally, these tests are accomplished at a certain point of time, often by in-lab enrichment of the bacterial sample.
Aptamers are well known in the field for their ability to bind to specific substances. Nucleic acid based aptamers are highly stable also. Aptamer specificity is often determined utilizing the systematic evolution of ligands by exponential enrichment (SELEX) method. This allows for high specificity to a wide variety of molecules. Aptamers are now gaining use as markers and linkers to cells. Aptamers are able to bind to the outer membrane proteins of cells and therefore act as markers and binders to the cell. (Joshua K. Herr et al., Aptamer-Conjugated Nanoparticles for Selective Collection and Detection of Cancer Cells, Analytical Chemistry, Vol. 78, No. 9, pp. 2918-2924, May 2006.)
Utilizing aptamer binding to Salmonella enterica has undergone proof of principle testing under Raghavendra Joshi et al. (Raghavendra Joshi et al., Selection, characterization, and application of DNA aptamers for the capture and detection of Salmonella enterica serovars, Molecular and Cellular Probes, Vol. 23, pp. 20-28, 2009). In those experiments, two highly specific 40-mer single DNA strand Salmonella enterica aptamers were discovered.
By utilizing the discovered two sequenced aptamers, Joshi et al, were able to utilize aptamer-infused magnetic particles to separate and concentrate Salmonella enterica bacteria in a sample, and thereby detect seven distinct serotypes of Salmonella enterica, with a detection sensitivity of about 10 CFU/gm.
U.S. Pat. No. 5,510,241 (“Thorns”) discloses a testing system for Salmonella bacteria, but does so utilizing monoclonal antibodies.
U.S. Pat. No. 5,582,981 (“Toole et al.”) discloses use of aptamer technology for binding to specific substances, but utilizes polymerase chain reaction. PCR testing requires a laboratory environment and a trained technician.
U.S. Pat. No. 5,635,617 (“Doran et al.”) discloses a specific target gene and protein of Salmonella bacteria; however, it does not apply this to a procedure for automated testing for the pathogen in food.
U.S. Pat. No. 5,712,17 (“Kouvonen et al.”) discloses a rapid immunoassay test strip that could be utilized for testing for pathogens, but does not disclose a way to do so in an automated way, and Kouvonen's method further requires a trained technician to accomplish the testing.
U.S. Pat. No. 5,840,867 (“Toole et al.”) discloses several specific aptamer sequences that may be utilized for targeting. However, it does not disclose a specific method for their use, nor does it disclose an aptamer specific to Salmonella enterica outer membrane proteins.
U.S. Pat. No. 6,680,377 B1 (“Stanton et al.”) discloses the composition of aptamers as beacons. Because this is not an electrochemical feedback system, it requires trained lab personnel and lab equipment. Also, this piece of prior art does not disclose a detection system for Salmonella enterica. 
What is needed in the field is a highly automated, accurate system that can be used outside of the laboratory environment, specifically at “Points-of-Inspection” such as ports, border check-points, and weighing stations along the normal paths of commerce by lay practitioners to accurately test for the presence of Salmonella in food samples in situ.